Antibiotics produced by Kibdelosporangium aridum shearer

ABSTRACT

A novel antibiotic complex of &#34;Vancomycin-like&#34; antibiotics, AAD 216 complex, is produced by the cultivation of a fermentation broth containing Kibdelosporangium aridum Shearer gen. nov., sp. nov. ATCC 39323 microorganisms in an aqueous nutrient medium under submerged aerobic conditions. The AAD 216 complex and its major bioactive components; AAD 216A, AAD 216B, and AAD 216C exhibit antibiotic activity and growth promotant activity.

SUMMARY OF THE INVENTION

This invention relates to new antibiotics of the vancomycin-class andthe production and recovery thereof. This invention also relates to thenew microorganism Kibdelosporangium aridum Shearer, gen, nov., sp. nov.SK&F-AAD 216 (ATCC 39323). More particularly, this invention relates toan antibiotic, designated herein AAD 216 complex, said complex beingproduced by cultivating K. aridum in an aqueous nutrient medium,containing assimilable sources of nitrogen and carbon, under submergedaerobic conditions until a substantial amount of AAD 216 complex isproduced by said microorganism in said medium and optionally recoveringAAD 216 complex from the culture medium.

Also provided for in this invention are the novel bioactive componentsof the AAD 216 complex; AAD 216A, AAD 216B and AAD 216C, which areprepared by the separation and isolation of the individual antibioticcompounds from the AAD 216 complex by chromatographic means. Theantibiotic, AAD 216 complex and its major bioactive components; AAD216A, AAD 216B and AAD 216C all exhibit antibacterial activity. The AAD216 complex, AAD 216A, AAD 216B and AAD 216C are also useful in animalhealth applications, such as a growth promotant and the treatment ofbovine mastitis.

DETAILED DESCRIPTION

The new antibiotics, AAD 216 complex and its major bioactive components;AAD 216A, AAD 216B and AAD 216C, are produced by the fermentation of anew microorganism Kibdelosporangium aridum Shearer gen. nov., sp. nov.SK&F-AAD 216. The above microorganism was isolated from a soil samplecollected in a desert area near Pima, Ariz. A culture of thebiologically pure microorganism has been deposited in the American TypeCulture Collection, Rockville, Md. as the type culture under accessionnumber ATCC 39323.

The AAD 216 complex refers to the mixture of the individual antibioticsproduced by the fermentation of K. aridum. As is readily understood bythose familiar with antibiotics fermentation processes, the ratios ofthe individual or factor antibiotics and their presence in the AAD 216complex will vary, depending on the conditions of the fermentationprocess. The AAD 216 complex may be recovered from the fermentationmedium by clarifying the whole fermentation broth by filtration and byprecipitating the crude AAD 216 complex at 0° to 10° C. withhydrochloric acid at a pH of 3. The precipitate is then dissolved inwater by adjusting the pH to about 6 to 8 and applied to a resin columnfrom which it is eluted with aqueous methanol. The resultant eluant islyophilized to yield the AAD 216 complex which is chromatographed toafford the enriched AAD 216 complex. Typically, the enriched AAD 216complex contains 40 to 85 percent by weight of a mixture of AAD 216A,AAD 216B and AAD 216C.

The enriched AAD 216 complex at a pH of about 6 which by analytical HPLCcontains by weight 29 percent AAD 216A, 10 percent AAD 216B and 10percent AAD 216C has the following characteristics:

(a) pale-white-yellow solid which decomposes at 300° to 350° C.;

(b) an appropriate elemental composition of 53.22 percent carbon, 6.14percent hydrogen, 3.73 percent nitrogen and 0.28 percent ash;

(c) an infrared spectrum in potassium bromide which exhibits peaks atthe following wave numbers in cm⁻¹ : 3400, 2920, 1660, 1600, 1510, 1460,1430, 1390, 1320, 1240, 1150, 1060 and 1020;

(d) an ultraviolet spectrum in acetonitrile:water (1:1) which exhibitsan absorption maximum at 280 nm under neutral and acid conditions withan E_(1%) =43.9 and at 301 nm under basic conditions with an E_(1%)=56.8;

(e) a positive reaction with periodate and negative reaction withninhydrin; and

(f) soluble in H₂ O, methanol, dimethylsulfoxide and dimethylformamideand insoluble in ethanol, acetonitrile, acetone, diethylether andaliphatic hydrocarbons.

The individual pure antibiotic components, AAD 216A, AAD 216B and AAD216C can be isolated from the AAD 216 complex utilizing preparative highpressure liquid chromatography (HPLC). Typically, the AAD 216 complex ischromatographed with a step gradient elution with 20 to 28 percentacetonitrile in 0.1N phosphate buffer at pH 6. The appropriatefractions, as determined by analytical HPLC, are combined and desaltedon a resin column and after lyophilization affords the desiredindividual antibiotic components, as pure AAD 216A, AAD 216B and AAD216C.

AAD 216A antibiotic at a pH of about 6 has the followingcharacteristics:

(a) pale white-yellow solid which decomposes at 300° to 350° C.;

(b) an empirical formula C₈₁ H₈₂ N₈ O₃₀ Cl₄

(c) an approximate elemental composition of 48.20 percent carbon, 5.01percent hydrogen, 5.21 percent nitrogen, 6.43 percent chlorine, nosulfur or organic phosphorus, when the water content was 10.80 percent;

(d) an infrared absorption spectrum in potassium bromide which exhibitspeaks at the following wave numbers in cm⁻¹ : 3400, 2920, 1660, 1600,1510, 1460, 1430, 1390, 1320, 1300, 1240, 1150, 1060 and 1020;

(e) a fast atom bombardment (FAB) mass spectrum with M+H at 1787 (majorcluster);

(f) an ultraviolet spectrum in acetonitrile:water (1:1) which exhibitsan absorption maximum at 280 nm under neutral and acidic conditions withan E_(1%) =51 and at 301 nm under basic conditions with an E_(1%) =73;

(g) a carbon magnetic resonance spectrum at 90.56 MHz in CD₃ OD:D₂O(1:9) at a pH of 8.5 which exhibits the following chemical shifts inpart per million (ppm) relative to TMS as standard: 177.7, 177.5, 175.5,174.6, 171.5, 170.8, 170.4, 170.2, 169.1, 161.8, 158.6, 157.9, 155.1,155.0, 152.5, 151.9, 151.6, 150.7, 146.0, 144.3, 141.7, 138.8, 138.3,136.0, 134.6, 134.5, 133.7, 130.8, 129.8, 129.4, 128.9 128.6, 127.4,127.0, 126.2, 125.6, 125.1, 122.7, 122.3, 120.9, 119.6, 118.3, 116.4,110.5, 109.6, 108.3, 104.2, 103.3, 103.1, 100.7, 98.1, 78.4, 74.4, 73.9,73.3, 72.0, 71.6, 71.3, 70.7, 67.3, 65.6, 63.6, 62.3, 61.6, 60.2, 56.8,56.3, 55.8, 54.9, 37.2, 32.7, 32.2, 29.8, 29.6, 29.5, 26.2, 23.0 and14.5;

(h) a specific rotation [α]_(D) ²⁵ =-66° (C=0.3 in H₂ O);

(i) positive reaction with periodate and negative reaction withninhydrin;

(j) soluble in H₂ O, methanol, dimethylsulfoxide, dimethylformamide andinsoluble in ethanol, acetonitrile, acetone, diethylether and aliphatichydrocarbons; and.

(k) pK_(a) values in acetonitrile:water (3:7) as follows: 3.0, 4.9, 7.4,8.4, 10.0 and 10.3 pK_(a) values above 10.3 not determined.

AAD 216B antibiotic at a pH of about 6 has the followingcharacteristics:

(a) pale white-yellow solid which decomposes at 300° to 350° C.,

(b) an empirical formula C₈₂ H₈₄ N₈ O₃₀ Cl₄

(c) an approximate elemental composition of 49.67 percent carbon, 5.07percent hydrogen, 5.19 percent nitrogen, 6.70 percent chlorine, nosulfur or organic phosphorus, when the water content is 10.8 percent;

(d) an infrared absorption spectrum in potassium bromide which exhibitspeaks at the following wave numbers in cm⁻¹ : 3400, 2920, 1660, 1600,1510, 1460, 1430, 1390, 1300, 1240, 1150, 1060 and 1020;

(e) a fast atom bombardment (FAB) mass spectrum with M+H at 1801 (majorcluster);

(f) an ultraviolet spectrum in acetonitrile; water (1:1) which exhibitsan absorption maximum at 280 nm under neutral and acidic conditions withan E_(1%) =55 and at 301 nm under basic conditions with an E_(1%) =72.5;

(g) a carbon magnetic resonance spectrum at 90.56 MHz in CD₃ OD:D₂O(1:9) at a pH of 8.5 which exhibits the following chemical shifts inparts per million (ppm) relative to TMS as standard: 177.9, 177.4,175.6, 174.4, 171.6, 170.9, 170.5, 170.4, 169.3, 162.4, 158.7, 158.1,155.2, 155.0, 152.6, 151.3, 150.7, 146.0, 144.3, 141.3, 138.8, 138.3,136.1, 134.7, 133.6, 130.7, 129.9, 129.8, 129.4, 129.0, 128.7, 127.7,127.5, 127.0, 126.3, 125.7, 124.7, 122.8, 122.3, 120.9, 119.9, 119.6,118.4, 116.7, 110.5, 109.6, 108.5, 104.2, 103.3, 103.1, 100.6, 98.1,78.5, 74.4, 73.9, 73.4, 72.1, 71.7, 71.2, 70.8, 67.3, 65.5, 63.7, 62.3,61.6, 60.3, 56.8, 56.2, 55.9, 55.0, 39.6, 37.3, 32.7, 30.2, 30.0, 29.7,28.4, 27.8, 26.3 and 23.3;

(h) a specific rotation [α]_(D) ²⁵ =-59° (C=1.0 in H₂ O);

(i) positive reaction with periodate and negative reaction withninhydrin;

(j) soluble in H₂ O, methanol, dimethylsulfoxide, dimethylformamide andinsoluble in ethanol, acetonitrile, acetone, diethylether, and aliphatichydrocarbons; and

(k) pK_(a) values in acetonitrile:water (3:7) as follows: 3.0, 4.5, 7.5,8.5 and 9.7 pK_(a) values above 9.7 not determined.

AAD 216C antibiotic at a pH of about 6 has the followingcharacteristics:

(a) pale white-yellow solid which decomposes at 300° to 350° C.;

(b) an empirical formula C₈₃ H₈₆ N₈ O₃₀ Cl₄

(c) an approximate elemental composition of 47.89 percent carbon, 5.09percent hydrogen, 4.95 percent nitrogen, 6.39 percent chlorine, 3.69ash, no sulfur or organic phosphorus, when the water content was 8.2percent;

(d) an infrared absorption spectrum in potassium bromide which exhibitspeaks at the following wave numbers in cm⁻¹ : 3400, 2920, 1660, 1600,1505, 1430, 1390, 1295, 1240, 1150, 1060 and 1020;

(e) a fast atom bombardment (FAB) mass spectrum with M+H at 1815 (majorcluster);

(f) an ultraviolet spectrum in acetonitrile:water (1:1) which exhibitsan absorption maximum at 280 nm under neutral and acid conditions withan E_(1%) =51 and at 301 nm under basic conditions with an E_(1%) =75;

(g) a carbon magnetic resonance spectrum at 90.56 MHz in CD₃ OD:D₂O(1:9) at a pH of 8.5 which exhibits the following chemical shifts inparts per million (ppm) using TMS as the internal standard: 177.9,177.2, 175.7, 173.6, 171.5, 170,8, 170.6, 170.2, 169.3, 158.6, 157.8,155.2, 154.9, 152.7, 151.9, 150.9, 146.0, 144.3, 141.3, 138.8, 138.4,136.0, 134.9, 134.7, 133.8, 130.8, 129.9, 129.8, 129.3, 129.1, 127.7,127.5, 127.0, 126.4, 125.3, 122.7, 121.0, 119.7, 118.3, 116.1, 110.4,109.6, 108.1, 104.1, 103.2, 101.5, 98.0 78.6, 74.6, 73.9, 73.4, 72.1,71.7, 71.3, 70.3, 67.3, 65.1, 63.7, 62.5, 61,6, 60.3, 56.8, 56.3, 55.3,54.9, 39.7, 37.4, 32.6, 32.3, 30.5, 30.4, 30.2, 29.9, 28.6, 28.1, 26.4,23.4 and 22.0;

(h) a specific rotation [α]_(D) ²⁵ =-50.6 (C=0.6 in H₂ O);

(i) positive reaction with periodate and negative reaction withninhydrin;

(j) soluble in H₂ O, methanol, dimethylsulfoxide, dimethylformamide andinsoluble in ethanol, acetonitrile, acetone, diethylether and aliphatichydrocarbons; and

(k) pK_(a) values in acetonitrile:water (3:7) as follows: 3.0, 4.2, 7.2,8.2, 9.9 and 10.3 pK_(a) values above 10.3 not determined.

The novelty of AAD 216 complex, and its major bioactive components, AAD216A, AAD 216B and AAD 216C, was confirmed by comparision with knownmembers of the vancomycin/ristocetin class of antibiotics. Specifically,the Rf values from thin layer chromatography against B. subtilis for AAD216A, AAD 216B and AAD 216C were compared to known antibiotics as shownbelow in TABLE I.

                  TABLE I                                                         ______________________________________                                                   TLC (Avicel).sup.1                                                            A       B          C                                               ______________________________________                                        A-35512B     0.14      0.25       0, 0.15                                     A-477        0.51      0.30, 0.40 0, 0.20                                                                       0.67, 0.78                                  Actaplanin   0.16      0.06       0.11                                        Actinoidins A                                                                               0.04,    0.18, 0.43 0.05                                        and B        0.24                                                             Avoparcin    0.15      0.13       0.07                                        LL-AM-374    0.08      0.17       0.05                                        Ristocetin   0.1       0, 0.10    0.04                                        Teichomycin A-2                                                                            0.34      0.12, 0.33,                                                                              0.77                                                               0.47                                                   Vancomycin   0.17      0, 0.35    0.1, 0.6                                    AAD 216A     0.35      0.33       0.83                                        AAD 216B     0.38      0.35       0.83                                        AAD 216C     0.44      0.34       0.82                                        ______________________________________                                         .sup.1 Rf values; detection: B. subtilis activity. Underlined value is        major active component                                                        A. nBuOH:HOAc:H.sub.2 O (4:1:5)                                               B. nPrOH:Petroleum Ether:conc. NH.sub.4 OH (4:1:2)                            C. nPrOH:H.sub.2 O (6:4)                                                 

Additionally, the retention times of AAD 216A, AAD 216B and AAD 216C inreverse phase high pressure liquid chromatography were compared to andfound to differ from those of the known antibiotics as shown below inTABLE II.

                  TABLE II                                                        ______________________________________                                                     Retention Time, Min.                                             Antibiotics    System A.sup.(a)                                                                        System B.sup.(b)                                     ______________________________________                                        AAD 216A       15.3      14.5                                                 AAD 216B       15.9      15.2                                                 AAD 216C       17.2      16.1                                                 A 35512B       5.2*      6.1*                                                 A 477          11.7*     8.9*                                                 Actaplanin     6.3*      7.5*                                                 Actinoidin A/B 5.5*      8.1*                                                 Avoparcin      5.8*      7.6*                                                 LL-AM-374      5.1*      6.8*                                                 Ristocetin     3.6       5.4                                                  Teichomycin A-2                                                                              13.6*     13.8*                                                Vancomycin     6.0       7.4                                                  ______________________________________                                         *major component in multicomponent mixture                               

.sup.(a) HPLC System A:

column: Ultrasphere ODS, 5 micron, 4.6×150 mm

solvent: 7-34% acetonitrile (7% for 1 minute, then ramp to 34% over 13minutes and hold at 34%) in pH 3.2, 0.1M phosphate.

flow: 1.5 ml/min

detection: uv 220 nm

.sup.(b) HPLC System B:

column: Ultrasphere ODS, 5 micron, 4.6×150 mm

solvent: 5-35% acetonitrile (5% for 1 minute, then ramp to 35% over 13minutes and hold at 35%) in pH 6.0, 0.025M phosphate

flow: 1.5 ml/min.

detection: uv 220 nm

Total hydrolysis of AAD 216A, AAD 216B and AAD 216C, individually, in 6Nhydrochloric acid at reflux for 18 hours yielded no common amino acidswhich could be detected in a standard amino acid analysis. The presenceof actinoidinic acid, which is common in the vancomycin class ofantibiotics, was confirmed in the hydrolysis products by high pressureliquid chromatography and FAB mass spectrum when compared to anauthentic sample. Further, hydrolysis of AAD 216A, AAD 216B and AAD216C, individually, in 1N hydrochloric acid at reflux for 4 hoursyielded mannose but not ristosamine, glucosamine or vancosamine.

Bioactive hydrolysis products of the compounds of this invention,including the aglycone of the following structural formula (I) ##STR1##are disclosed and claimed in U.S. Pat. No. 4,521,335.

THE MICROORGANISM

Stock cultures of Kibdelosporangium aridum SK&F-AAD 216 (ATCC 39323)were maintained on thin potato-carrot or oatmeal agar. Morphologicalobservations were made on plates of water agar, thin potato-carrot agar,oatmeal agar and soil extract agar. Inoculum for the biochemical andphysiological tests was prepared by adding the contents of a frozen vialof vegetative culture to a flask of glucose-yeast extract broth whichwas placed on a rotary shaker at 28° C. 250 RPM for three to six days.The culture was harvested by centrifugation and washed three times withsterile distilled water. Incubation temperature for the biochemical andphysiological tests was 28° C. Readings of the results were made atvarious times up to 21 days for the plate media. Most of the tubed mediawere read at various times up to 28 days. However, the tests fordecomposition of urea, allantoin and hippurate, as well as the tests forreduction of nitrates, were read for six weeks.

Susceptibility of K. aridum to antibiotics was examined by placing BBLsusceptibility disks on nutrient agar plates seeded with K. aridum as anoverlay. Diameters of the zones of inhibition were measured afterincubation for one week at 28° C.

Morphology. K. aridum is a filamentous organism that forms a myceliumdifferentiated into: (1) a substrate mycelium that penetrates the agarand forms a compact layer on top of the agar, and (2) an aerial myceliumthat bears chains of conidia and/or sporangium-like structures. Nomotile elements were observed in the aerial or substrate mycelium. Onmany media, K . aridum produces characteristic crystals in the agar.

Substrate Mycelium. K. aridum produces a well developed, non-fragmentingsubstrate mycelium. The long branching hyphae are septate and about 0.4μm-1.0 μm in diameter. Present on the substrate mycelium are specializedstructures which consist of dichotomously branched, septate hyphaeradiating from a common stalk.

Aerial Mycelium. The aerial mycelium of K. aridum, produces chains ofrod-shaped, smooth walled spores which are irregular in length (0.4μm×0.8 μm-2.8 μm). These spore chains are usually very long with morethan 50 spores per chain, but a few short chains of ten spores or lessare also usually present. The spore chains may be born apically or onshort lateral branches.

On most media, the aerial mycelium of K. aridum also producessporangium-like structures. These may be born apically or on shortlateral branches. Sporangium-like structures and chains of spores may beborn on the same aerial hyphae. At maturity these sporangium-likestructures are round, approximately 9 μm-22 μm in diameter, and consistof septate, branches hyphae embedded in an amorphous matrix which issurrounded by a well-defined wall. When placed on agar, thesesporangium-like structures germinate directly with the production of oneor more germ tubes.

Chemotaxonomy. Purified cell wall preparations of K. aridum analyzed bythe method of Becker [Becker et al. Appl. Microbiol. 12, 421-23 (1964)]contained the meso-isomer of 2,6-diaminopimelic acid, alanine, glutamicacid, glucosamine and muramic acid as well as galactose and a very minoramount of arabinose. Whole-cell hydrolysates analyzed by the method ofLechevalier [Lechevalier, M. P., J. Lab. Clin. Med. 71, 934-44 (1968)]contained galactose, glucose, mannose, ribose, rhamnose and arabinose;traces of madurose may also be present. No mycolic acids of any typewere present in the cell extracts analyzed for lipid patterns by themethod of Lechevalier [Lechevalier et al., Can. J. Microbiol. 19, 965-72(1973)]. The phospholipids present were phosphatidyl ethanolamine,phosphatidyl methylethanolamine, diphosphatidyl glycerol, phosphatidylinositol and phosphatidyl inositol mannosides. Thus K. aridum has a TypeIV cell wall with a whole-cell sugar pattern of Type A with traces ofmadurose [Lechevalier et al., Int. J. Syst. Bacteriol. 20, 435-43(1970)] and a phospholipid pattern of type PII with phosphatidylmethylethanolamine [Lechevalier et al., Biochem. System. Ecol. 5, 249-60(1977)].

Physiological and Biochemical Characteristics. K. aridum is grampositive and not acid-fast. No growth takes place under anaerobicconditions. Temperature range for growth is 15° C. to 42° C. with atrace of growth at 45° C.; no growth occurs at 10° C. or 50° C. Hydrogensulfide is produced. Milk is peptonized. Gelatin is both hydrolyzed andliquified. Nitrate is not reduced to nitrite. Melanin pigments areproduced. Casein, L-tyrosine, hypoxanthine, quanine, elastin andtestosterone are hydrolyzed but adenine, xanthine and cellulose (Avicel)are not. Catalase and phosphatase are produced. Urea, esculin andhippurate are decomposed; tests for allantoin decomposition are weaklypositive. There is no growth in 8% NaCl; growth in 5% to 7% NaCl isinconsistent. No growth occurs in lysozyme broth. Acid is produced fromL-arabinose, D-cellobiose, dextrin, dextrose, D-fructose, glycerol,glycogen, D-galactose, i-inositol, lactose, D-mannitol, D-mannose,α-methyl-D-glucoside, α-methyl-D-mannoside, melibiose, D-melezitose,raffinose, rhamnose, D-ribose, sucrose, trehalose, D-xylose and maltose.No acid is produced from dulcitol, i-erythritol, inulin, D-sorbitol, orL-sorbose. Citrate, malate, succinate, oxalate, lactate, acetate,pyruvate, propionate and formate are utilized; benzoate and tartrate arenot.

Susceptibility to Antibiotics. By the diffusion method, K.. aridum wasresistant to disks impregnated with gentamicin (10 μg), tobramycin (10μg), streptomycin (10 μg), vancomycin (30 μg), penicillin (10 units),bacitracin (2 units), lincomycin (2 μg), clindamycin (2 μg), andcephalothin (30 μg). Chlortetracycline (5 g) produced 18 mm zones ofinhibition; tetracycline (5 μg) 11-12 mm zones; rifampin (5 μg) 11-15 mmzones; novobiocin (5 μg) 27-28 mm zones. All zones of inhibitioncontained at least a few resistant colonies.

Description of K. aridum on Various Media

All cultures were incubated at 28° C. in closed petri dish cans andobserved at intervals up to 21 days. The colors of the culture werechosen by comparison with color chips from either the ISCC-NBS CentroidColor Charts or the Dictionary of Color [Maerz, A. and M. R. Paul 2nd.ed. New York: McGraw Hill Book Co., Inc. (1950)].

Yeast Extract-Malt Extract Agar--Growth excellent; vegetative growthgrayish yellow brown; aerial mycelium, none to very sparse, white, sporechains and sporangium-like structures present; yellow brown solublepigment; characteristic crystals present in agar.

Oatmeal Agar--Growth good; vegetative growth off-white to yellow brown;aerial mycelium sparse, white, numerous spore chains and sporangium-likebodies present; no soluble pigment; characteristic crystals present inagar.

Glycerol-Asparagine Agar--Growth fair to good; vegetative growth paleyellow brown; aerial mycelium sparse to moderate, white, a few sporechains but few, if any, sporangium-like structures present; pale grayishyellow brown soluble pigment; characteristic crystals present in agar.

Inorganic-Salts Starch Agar--Growth good; vegetative growth off-white toyellow brown; aerial mycelium sparse, white, spore chains andsporangium-like structures present; no soluble pigment; characteristiccrystals present in agar.

Czapek-Sucrose Agar--Growth good; vegetative growth off-white to yellowbrown; aerial mycelium sparse, white, spore chains and sporangium-likestructures present; pale grayish yellow to pale yellow brown solublepigment present; characteristic crystals present in agar.

Bennett's Agar--Growth good to excellent; vegetative growth grayishyellow brown; aerial mycelium, none to sparse, white, spore chains andsporangium-like structures present; yellow brown soluble pigment; nocrystals detected in agar.

Nutrient Agar--Growth fair to good; vegetative growth yellow brown;aerial mycelium sparse to moderate, white, few spore chains and fewsporangium-like structures present; yellow brown soluble pigment;characteristic crystals variably present in agar.

Thin Potato-Carrot Agar--Growth fair, relatively flat; vegetative growthoff-white to yellow brown; aerial mycelium sparse to moderate, white,numerous spore chains and sporangium-like structures present; no solublepigment; no crystals detected in agar.

Peptone-Yeast Extract Iron Agar--Growth good; vegetative growth bronzebrown (Maerz & Paul 16C9); aerial mycelium, none; dark brownish blacksoluble pigment; no crystals detected in agar.

Starch-Casein Nitrate Agar--Growth good; vegetative mycelium off-whiteto yellow brown; aerial mycelium sparse, white, spore chains andsporangium-like structures present; pale yellow brown soluble pigmentvariably present; characteristic crystals present in agar.

Yeast Extract-Glucose Agar--Growth good to excellent; vegetative growthdark yellow brown to grayish yellow brown; aerial mycelium, nonevisible, under 400X sparse spore chains but no sporangium-likestructures present; dark yellow brown soluble pigment; characteristiccrystals present in agar.

A comparison of the description of K. aridum with the descriptions ofactinomycetes listed in Bergey's Manual of Determinative Bacteriology,the Approved List of Bacterial Names and other recent taxonomicliterature indicates that K. aridum differs significantly frompreviously described species of actinomycetes and cannot be accommodatedin any of the previously described genera of the actinomycetes.Sporangia, where present in other actinomycete genera, are true sporevesicles; at maturity they contain aplanospores or zoospores which areeventually released by rupture or dissolution of the sporangial wall.Despite extensive observation and manipulation, the release of sporesfrom the sporangium-like structures of K. aridum was never observed.When placed on agar, the sporangium-like structures of K. aridumgerminate by the production of one or more germ tubes directly from thesporangium-like structure.

Type culture ATCC 39323 is hereby described as a species of a new genusKibdelosporangium aridum, (from kibdelos, Gr. adj., false, ambiguous;spora Gr. n., a seed; angium Gr. n., a vessel); the specific epithet,aridum, (aridus Gr. n., L. adj., dry, arid) refers to the desert soilfrom which the culture was isolated.

PREPARATION OF THE AAD 216 COMPLEX

The AAD 216 complex may be produced by cultivating a strain ofKibdelosporangium having the characteristics of ATCC 39323 or an activemutant or derivative thereof, obtained by procedures known to the art,under submerged aerobic conditions in an aqueous nutrient medium. Theorganism is grown in a nutrient medium containing an assimilable carbonsource, for example an assimilable carbohydrate. Examples of suitablecarbon sources include sucrose, lactose, maltose, mannose, fructose,glucose, and soluble starch. The nutrient medium should also contain anassimilable nitrogen source such as fish meal, peptone, soybean flour,peanut meal, cotton seed meal or corn steep liquor. Nutrient inorganicsalts can also be incorporated in the medium. Such salts may compriseany of the usual salts capable of providing sodium, potassium, ammonium,calcium, phosphate, sulfate, chloride, bromide, nitrate, carbonate orlike ions.

Production of the AAD 216 complex can be effected at any temperatureconducive to satisfactory growth of the organism, e.g., 15°-42° C., andis conveniently carried out at a temperature of about 25° to 28° C.

The medium normally is neutral, but the exact pH can be varied between5.0 and 9.0 depending on the particular medium used.

The fermentation may be carried out in Erlenmeyer flasks or inlaboratory or industrial fermentors of various capacities. When tankfermentation is to be used, it is desirable to produce a vegetativeinoculum in a nutrient broth by inoculating a small volume of theculture medium with the vegetative cells of the organism. Afterobtaining an inoculum in this manner, it is transfered aseptically tothe fermentation tank medium for large scale production of theantibiotics. The medium used for the vegetative inoculum can be the sameas that employed for larger fermentations, although other media can beemployed.

As is customary in aerobic submerged culture processes, sterile air issparged through the culture medium. Agitation may be maintained by meansof agitators generally familiar to those in the fermentation industry.

In general, optimum production of the complex is achieved afterincubation periods of about 144-186 hours in stir-jar fermentors or tankfermentors. The course of the fermentation can be followed by analyticalHPLC.

The AAD 216 complex so produced contains the novel bioactive componentsor individual antibiotic factors, AAD 216A, AAD 216B and AAD 216C whichcan be isolated as described above.

BIOLOGICAL ACTIVITY DATA

The in vitro minimum inhibitory concentration (MIC) of the AAD 216complex, enriched AAD 216 complex, AAD 216A, AAD 216B, AAD 216C andvancomycin were determined for a number of microorganisms using thestandard microtiter assay procedures. The results are shown in thefollowing Tables A-E.

                  TABLE A                                                         ______________________________________                                        Antimicrobial Spectrum                                                                   MIC in μg/ml                                                                 AAD                                                                           216     AAD     AAD   AAD   Van-                                              Com-    216     216   216   comy-                                Test Organism                                                                              plex    A       B     C     cin                                  ______________________________________                                        Staph. aureus HH127                                                                        6.3     3.1     3.1   3.1   1.6                                  Staph. aureus                                                                              12.5    3.1     3.1   3.1   1.6                                  SK&F 910                                                                      Strep. faecilis is HH34358                                                                 1.6     SK&F 0.4                                                                              0.4   0.8   3.1                                  Proteus mirabilis                                                                          >100    >100    >100  >1.00  100                                 SK&F 444                                                                      E. coli 12140                                                                              >100    >100    >100  >100   100                                 (SK&F 809)                                                                    Kleb. pneumoniae                                                                           >100    >100    >100  >100  >100                                 4200 (SK & F 798)                                                             Pseudomonas aerugin-                                                                       >100    >100    >100  >100  >100                                 OSA HH63                                                                      Serratia marcesens                                                                         >100    >100    >100  >100  >100                                 ATCC 13880                                                                    Proteus morgani                                                                            >100    >100    >100  >100  >100                                 SK&F 179                                                                      Providencia SK & F 276                                                                     >100    >100    >100  >100  >100                                 Enterobacter cloacae                                                                       >100    >100    >100  >100  >100                                 HH31254                                                                       Salmonella   >100    >100    >100  >100    25                                 gallinarum BC595                                                              Staph. epidermidis                                                                          25     6.3     6.3   6.3   1.6                                  SK & F 2479                                                                   Listeria monocytogenes                                                                     3.1     0.8     0.4   ˜0.4                                                                          1.6                                  SK & F 2255                                                                   Staph. epidermidis                                                                          100      50      25    25  1.6                                  SK&F 651                                                                      ______________________________________                                    

                  TABLE B                                                         ______________________________________                                        (Methicillin Sensitive)                                                              MIC in μg/ml                                                                 AAD      AAD     AAD   AAD                                           Test     216      216     216   216                                           Organism Complex  A       B     C     Vancomycin                              ______________________________________                                        Staph. aureus                                                                          12.5     1.6-3.1 3.1   3.1   1.6                                     HH127                                                                         Staph. aureus                                                                          12.5     3.1-6.3 6.3   12.5  1.6                                     SK&F 674                                                                      Staph. aureus                                                                          12.5     3.1     6.3   6.3   1.6                                     SK&F 910                                                                      Staph. aureus                                                                          12.5     3.1     3.1   3.1   1.6                                     SK&F 1761                                                                     Staph. aureus                                                                          25       6.3     6.3   6.3   1.6                                     SK&F 2593                                                                     Staph. aureus                                                                          12.5     3.1     3.1   6.3   1.6                                     SK&F 2666                                                                     Staph. aureus                                                                          25       6.3     3.1   6.3   1.6                                     SK&F 2677                                                                     Staph. aureus                                                                          25       6.3     6.3   6.3   1.6                                     SK&F 2678                                                                     Staph. aureus                                                                          12.5     3.1-6.3 6.3   6.3   1.6                                     SK&F 2680                                                                     Staph. aureus                                                                          25       3.1-6.3 6.3   3.1   1.6                                     SK&F 2682                                                                     Staph. aureus                                                                          25       3.1-6.3 6.3   6.3   1.6                                     SK&F 2736                                                                     Staph. aureus                                                                          50       12.5    6.3   12.5  1.6                                     SK&F 2776                                                                     Staph. aureus                                                                          12.5     3.1     3.1   3.1   0.8                                     SK&F 2777                                                                     Staph. aureus                                                                          6.3      1.6     1.6   1.6   1.6                                     SK&F 2613                                                                     Staph. aureus                                                                          12.5     3.1-6.3 6.3   6.3   3.1                                     SK7F 2615                                                                     ______________________________________                                    

                  TABLE C                                                         ______________________________________                                        (Methicillin Resistant)                                                              MIC in μg/ml                                                                 AAD      AAD     AAD   AAD                                           Test     216      216     216   216                                           Organism Complex  A       B     C     Vancomycin                              ______________________________________                                        Staph. aureus                                                                          50       12.5    6.3   12.5  1.6                                     SK&F 675                                                                      Staph. aureus                                                                          12.5     6.3     3.1   6.3   1.6                                     SK&F 2612                                                                     Staph. aureus                                                                          6.3      3.1     3.1   3.1   1.6                                     SK&F 2614                                                                     Staph. aureus                                                                          25       6.3-12.5                                                                              6.3   6.3   0.8                                     SK&F 2616                                                                     Staph. aureus                                                                          25       6.3     12.5  12.5  3.1                                     SK&F 2620                                                                     Staph. aureus                                                                          25       6.3     6.3   6.3   1.6                                     SK&F 2621                                                                     Staph. aureus                                                                          25       6.3-12.5                                                                              6.3   6.3   1.6                                     SK&F 2594                                                                     Staph. aureus                                                                          25       6.3-12.5                                                                              6.3   6.3   1.6                                     SK&F 2589                                                                     Staph. aureus                                                                          25       6.3-12.5                                                                              3.1   6.3   1.6                                     SK&F 2590                                                                     Staph. aureus                                                                          25       6.3     6.3   6.3   1.6                                     SK&F 2591                                                                     Staph. aureus                                                                          50       6.3     12.5  12.5  3.1                                     SK&F 2592                                                                     Staph. aureus                                                                          25       6.3-12.5                                                                              6.3   6.3   ˜1.6                              SK&F 2595                                                                     Staph. aureus                                                                          25       6.3-12.5                                                                              6.3   6.3   3.1                                     SK&F 2596                                                                     Staph. aureus                                                                          25       6.3-12.5                                                                              6.3   12.5  3.1                                     SK&F 2597                                                                     ______________________________________                                    

                  TABLE D                                                         ______________________________________                                        (Anaerobes)                                                                           MIC in μg/ml                                                                 AAD      AAD       AAD   AAD   Van-                                           216      216       216   216   comy-                                Test Organism                                                                           Complex  A         B     C     cin                                  ______________________________________                                        Bacteroides                                                                             >32      32        32    32    32                                   fragilis ATCC 25285                                                           B. fragilis H145                                                                        >32      16        16    8     32                                   B. fragilis SK & F 3060                                                                 >32      32        16    16    32                                   B. loeochii SK & F 3087                                                                 >32      8-16      16    4     32                                   B. thetaiotamicron                                                                      >32      >32       32    32    32                                   SK & F 3089                                                                   Fusobacterium                                                                           >32      32        32    32    32                                   nucleatum ATCC 25586                                                          Clostridium                                                                             ≦0.016                                                                          ≦0.016                                                                           0.125 ≦ 0.016                                                                      0.5                                  perfringens                                                                   MCP-1                                                                         C. perfringens                                                                          ≦0.016                                                                          ≦0.016                                                                           0.125 ≦ 0.016                                                                      0.5                                  MCP-2                                                                         C. perfringens                                                                          0.25     0.03-0.063                                                                              0.125 0.125 1.0                                  ATCC 19408                                                                    Clastridium                                                                             1.0      0.25      0.25  0.5   2                                    difficile SK & F 3062                                                         C. difficile                                                                            1.0      0.5       0.25  0.5   4                                    SK&F 3065                                                                     C. difficile                                                                            ≦0.016                                                                          0.125-0.25                                                                              0.25  0.031 2                                    SK&F 3091                                                                     C. difficile                                                                            1.0      0.125     0.25  0.25  2                                    SK&F 3092                                                                     C. difficile                                                                            1.0      0.25      2     0.25  2                                    SK&F 3096                                                                     C. difficile                                                                            ≦0.016                                                                          ≦0.016                                                                           2     0.031 2                                    SK&F 3098                                                                     ______________________________________                                    

                  TABLE E                                                         ______________________________________                                                  MIC in μg/ml                                                                 Enriched                 Vanco-                                   Test Organism                                                                             AAD 216 Complex                                                                             AAD 216A   mycin                                    ______________________________________                                        Staph. aureus                                                                             6.3           6.3        3.1                                      HH 127                                                                        Staph. aureus                                                                             ˜6.3    6.3        3.1                                      SK&F 910                                                                      Staph. aureus209P                                                                         1.6           1.6        1.6                                      209P                                                                          Staph. aureus                                                                             100           100        >100                                     209P-Mutant                                                                   Staph. aureus                                                                             6.3           3.1        1.6                                      SK&F 674                                                                      Staph. aureus                                                                             ˜100    ˜100 >100                                     SK&F 674-P6                                                                   Mutant                                                                        Staph. aureus                                                                             12.5          12.5       3.1                                      SK&F 675                                                                      Staph. epidermidis                                                                         25            25        6.3                                      SK&F 2479                                                                     Staph. epidermidis                                                                        100           100        6.3                                      SK & F 2683                                                                   Staph. epidermidis                                                                        100           100        6.3                                      SK&F 651                                                                      Staph. epidermidis                                                                        100           100        6.3                                      SK & F 2265                                                                   Strep. faecalis HH 34358                                                                  0.4           1.6        SK&F 6.3                                 Strep. faecalis                                                                           0.4           0.8        3.1                                      SK & F 657                                                                    Listeria    0.8           1.6        3.1                                      monocytoqenes                                                                 SK & F 2255                                                                   E. coli 12140                                                                             >100          >100       >100                                     (SK & F 809)                                                                  Salmonella  >100          >100       >100                                     gallinarum                                                                    BC595                                                                         ______________________________________                                    

The in vivo activity of the AAD 216A, AAD 216B, AAD 216C and vancomycin,measured as ED₅₀, was demonstrated against intraperitoneal s.c.infections with 46.8 LD₅₀ 's of Staph. aureus HH 127 in mice bytreatments with the antibiotics 1 and 5 hours post infections. The ED₅₀'s were as follows: AAD 216A, 5.0 mg/kg; AAD 216B, 5.0 mg/kg; AAD 216C,7.5 mg./kg; vancomycin, 1.76 mg/kg.

The antibiotic compounds of the present invention including AAD 216complex and its major bioactive components; AAD 216A, AAD 216B and AAD216C and mixtures thereof, exhibit antibacterial activity. The inventionincludes within its scope pharmaceutical compositions containing atleast one of the above-mentioned antibiotic compounds and apharmaceutically acceptable carrier. The compositions may also containother active antibacterial agents. The compositions may be made up inany pharmaceutical form appropriate for the route of administration inquestion. Such compositions are exemplified by solid compositions fororal administration, such as tablets, capsules, pills, powders andgranules; liquid compositions for oral administration such as solutions,suspensions, syrups and elixers; and preparations for parenteraladministration such as sterile solutions, suspensions or emulsions.

For use as an antibacterial agent, the compositions are administered sothat the concentration of the active ingredients is greater than theminimum inhibitory concentration for the particular organism treated.

The activity of the AAD 216 complex, and its components; AAD 216A, AAD216B and AAD 216C was demonstrated in vitro against a total of 58 bovinemastitis isolates using the conventional agar dilution method todetermine minimum inhibitory concentrations (MICs). The MICs for the AAD216 complex, AAD 216A, AAD 216B and AAD 216C ranged from 0.25 to >128μg/ml, 0.13 to >128 μg/ml, 0.06 to >128 μg/ml and 0.06 to >128 μg/mlrespectively. In comparison, vancomycin has MICs for the samemicroorganisms ranging from 0.25 to >128 μg/ml.

GROWTH PROMOTANT ACTIVITY

The growth promotant activities of the AAD 216 complex, and itscomponents; AAD 216A, AAD 216B and AAD 216C were determined in a swinein vitro model to predict utility in monogastric animals, such as swineand poultry; a rumen in vitro model to predict utility in beef, dairyand sheep production and in vivo in a chick growth model.

Swine in vitro model

A Yorksire barrow is surgically prepared either with an ileal cannula,which is placed 15 cm. from the ileo-cecocolic junction, or a cecalcannula, which placed midway between the apex and origin of the cecum.The animal is fed 4 times daily to restrict intake to 4.5% of bodyweight in a 30 kg animal or 2.5% of body weight in a 100 kg animal. Theswine grower ration is:

    ______________________________________                                                          (% w/w)                                                                              (lbs/ton)                                            ______________________________________                                        Medium ground shelled corn                                                                        70.60    1412                                             Soybean meal, 44%   22.00    440                                              Dehydrated alfalfa meal, 17%                                                                      4.50     90                                               Calcium propionate  0.15      3                                               Vitamin/mineral premix                                                                            2.75     55                                               ______________________________________                                    

Sampling of the material, via the cannula, begins 150-180 minutesfollowing the first morning feeding and continues any time from 30-120minutes thereafter, depending on the quantity of material needed. Thesample is maintained in crushed ice, no cooler than 5° C., and is gassedcontinuously with carbon dioxide. The collected material is filtered.The filtrate is the inoculum used for incubations of the test andcontrol samples. The gassed inoculum, 2.25 ml, is placed in each of 10gassed test tubes, each containing 0.75 ml of a nutrient solution and0.5 mg of each test compound. Four blank control tubes, along with thetest compound tubes, are incubated 5 hours at 37° C. with agitation.Four more killed tubes are included which are not incubated.

The tubes are each treated with 0.60 ml of a 25% solution ofmetaphosphoric acid, then, stored at -4° C. until analysis. Samples arethawed and centrifuged for 25 minutes at 20,000 r.p.m. The supernatantliquid is decanted, sampled for gas chromatography and automaticanalysis. The results are fed into a computer for finishing to givefigures in which the blank control value is 100%. Virginiamycin andvancomycin are used as positive controls.

    ______________________________________                                                   VFA*     LYS*     GLU*                                                        (% Con-  (% Con-  (% Con-                                                                              LAC*                                      Compound (ppm)                                                                           trol)    trol)    trol)  (% Control)                               ______________________________________                                        Virginiamycin                                                                 (166.67)    93      163      197    81                                         (16.67)   130      127      191    76                                         (1.67)    248      82       182    70                                        Vancomycin                                                                    (166.67)   250      48       190    64                                         (16.67)   280      42       189    59                                         (1.67)     92      83       100    99                                        AAD 216 Complex                                                               (666.67)** 267      44       189    58                                        (166.67)   345      50       195    51                                         (66.67)   390      53       188    46                                         (16.67)   124      81       113    93                                         (6.67)     90      101       96    100                                       AAD 216A                                                                      (166.67)   326      63       187    58                                         (16.67)   379      50       191    47                                         (1.67)    101      94        97    99                                        AAD 216B                                                                      (166.67)   290      50       190    56                                         (16.67)   365      55       184    47                                         (1.67)    102      98        97    98                                        AAD 216C                                                                      (166.67)   294      42       191    57                                         (16.67)   398      53       188    45                                         (1.67)    101      95        96    98                                        ______________________________________                                         *VFA refers to the total of volatile fatty acids, namely acetate,             propionate, isobutyrate, butyrate, isovalerate and valerate. LYS is           lysine, GLU is glucose and LAC is Llactic acid.                               **AAD 216 complex contains 25% of a mixture of AAD 216A, AAD 216B and AAD     216C.                                                                    

Rumen in vitro model

The protocol for the rumen in vitro model is analogous to the protocolfor the swine in vitro model with the following modifications:

(1) A 400 kg steer is surgically prepared with a rumen cannula.

(2) The animal is fed one time a day with the following ration:

    ______________________________________                                                           % w/w                                                      ______________________________________                                        Finished Feed                                                                 Cottonseed hulls     44.0                                                     Cracked corn         22.0                                                     Alfalfa Hay 1"       20.0                                                     Pellet Supplement*   10.0                                                     Liquid Molasses      4.0                                                                           100.0                                                    *Pellet Supplement                                                            Soybean Oil Meal (50% protein)                                                                     50.0                                                     Medium Ground Corn   32.5                                                     D/Calcium Phosphate  6.50                                                     Plain salt           2.50                                                     Ground limestone (Thomasville)                                                                     3.50                                                     Urea                 2.50                                                     Vitamin A & D.sub.2 Premix**                                                                       2.50                                                                          100.00                                                   **Vitamin A & D.sub.2 Premix                                                  Vitamin A (30,000 IU/gm)                                                                           5.87                                                     Vitamin D.sub.2 (16,000,000 IU/lb)                                                                 0.50                                                     Fine ground corn     93.63                                                                         100.00                                                   ______________________________________                                    

(3) Manipulation of VFA production is described as the production ratioof propionate as a percentage of total VFA produced.

(4) Sampling of the material, via the cannula, is at 120 minutes postfeeding.

    ______________________________________                                                                             Propionate                                           VFA*     LYS*     GLU*   %                                                    (% Con-  (% Con-  (% Con-                                                                              (% Con-                                  Compound (ppm)                                                                            trol)    trol)    trol)  trol)                                    ______________________________________                                        Vancomycin                                                                    (50.0)      107      97       189    127                                       (5.0)      108      85       165    130                                       (0.5)       97      83        17    105                                      Avoparcin                                                                     (50.0)      113      97       141    132                                       (5.0)      105      85       166    121                                       (0.5)      103      96       116    106                                      Monensin Sodium                                                               (50.0)      109      147       0     156                                       (5.0)      104      141      126    149                                       (0.5)       94      109       11    119                                      AAD 216 Complex                                                               (200.0)**   118      116       78    159                                       (50.0)**   117      101       11    159                                       (20.0)**   115      95       193    130                                       (2.0)**    101      92        60    102                                      AAD 216A                                                                      (50.0)      111      114      153    146                                       (5.0)       93      90        28    135                                       (0.5)      94       96       121    105                                      AAD 216B                                                                      (50.0)      111      124       20    148                                      (5.0)       121      107       0     141                                      (0.5)       100      103       45    103                                      AAD 216C                                                                      (50.0)      101      110       0     151                                       (5.0)      111      96        45    136                                       (0.5)       83      103       29    120                                      ______________________________________                                         *VFA refers to the total of volatile fatty acids, namely acetate,             propionate, isobutyrate, butyrate, isovalerate and valerate. LYS is           lysine, and GLU is glucose.                                                   **AAD 216 complex contains 25% of a mixture of AAD 216A, AAD 216B and AAD     216C.                                                                    

Chick Growth Study

One day old broiler chicks, selected for weight, health and sex, arehoused in an environmentally controlled room with temperature at 80° F.and humidity at 40%. Chicks are fed ad libitum. Water is offered adlibitum. A rye or corn basal ration is fed during the acclimation period(days 1 and 2), then, mixed with the compound under test or controlconditions on days 3-17. Either 8 pens (64 chicks) or 16 pens (128chicks) are used for each test or control group.

    ______________________________________                                        Basal Rye Diet                                                                Diet Ingredients     (% w/w)  (lbs/ton)                                       ______________________________________                                        Ground Rye (fine grind)                                                                            54.4     1088                                            Soybean Meal (49% protein)                                                                         27       540                                             Meat & Bone meal (50% protein)                                                                     10       200                                             Dehydrated Alfalfa meal                                                                            1.25     25                                              Fat, animal          4        80                                              Dried Whey (or lactose)                                                                            1        20                                              Ground Limestone     0.67     13.4                                            Dicalcium Phosphate  0.50     10                                              Iodized salt         0.23     4.6                                             Vitamin premix       0.175    *                                               Trace mineral premix 0.25     5                                               DL methionine (98%)  0.45     9                                               Choline Chloride (50% aqueous sol.)                                                                0.150**  3                                               ______________________________________                                         *Vitamin premix will be mixed into diets when test chemicals are added.       87.5 g vitamin premix/49,912.5 g of basal rye diet.                           **Since choline is added as a 50% aqueous solution, percentage in diet is     doubled.                                                                 

    __________________________________________________________________________              Dose # of                                                                             Weight  Feed/Gain   # of                                              PPM  Reps                                                                             Day 10                                                                            Day 17                                                                            3-10                                                                              10-17                                                                             3-17                                                                              D. Cks                                  __________________________________________________________________________                      % of Control                                                Virginiamycin                                                                           10.0 8  103.8                                                                             116.0                                                                             97.4                                                                              83.6                                                                              90.4                                                                              2                                       Virginiamycin                                                                           50.0 8  105.0                                                                             121.3                                                                             93.2                                                                              74.6                                                                              83.2                                                                              1                                       AAD 216 Complex*                                                                        40.0 8  107.7                                                                             132.4                                                                             90.9                                                                              69.6                                                                              79.6                                                                              2                                       AAD 216 Complex*                                                                        160.0                                                                              8  106.1                                                                             117.1                                                                             91.0                                                                              80.1                                                                              85.3                                                                              3                                                         GRAMS   GRAMS/GRAM                                          Control   0.0  8  164.2                                                                             280.4                                                                             1.577                                                                             2.815                                                                             2.209                                                                             2                                       Rye                                                                           Historic Control  169.1                                                                             298.4                                                                             1.566                                                                             2.563                                                                             2.080                                       C.V. Among                                                                    Pens-3.71%                                                                    __________________________________________________________________________     *AAD 216 complex contains 25% of a mixture of AAD 216A, AAD 216B and AAD      216C.                                                                    

The feed compositions of this invention comprise the normal feed rationsof the meat and milk producing animals supplemented by a quantity of anactive ingredient selected from the group consisting of AAD 216 complex,AAD 216A, AAD 216B, AAD 216C or a mixture thereof which is effective forimproving the growth rate and feed efficiency of the animals but whichis not toxic or noxious to a degree that the animals will reduceingestion of the ration. The quantity of the active ingredient willvary, as is known to the art, with factors such as the cost of theingredient, the species and the size of animal, the relative activity ofthe compound of formula I or the type of feed ration used as the basalfeed.

Representative feed rations for swine and poultry are as follows:

A swine ration for growing hogs of 40-100 pounds body weight is preparedusing the following formula:

    ______________________________________                                        Corn, ground       78.15%                                                     Soybean oil meal, 44%                                                                            17.0%                                                      Meat scraps, 50%   3.0%                                                       Oyster shell flavor                                                                              0.4%                                                       Bone meal          0.5%                                                       Zinc oxide         0.01%                                                      Vitamin A, B, B.sub.12 & D                                                                       optional                                                   supplement                                                                    ______________________________________                                    

A chicken ration for broilers is prepared using the following formula:

    ______________________________________                                        Yellow corn meal  67.35%                                                      Soybean oil meal  24.00%                                                      Menhaden fish meal                                                                              6.00%                                                       Steamed bone meal 1.00%                                                       Ground limestone  1.00%                                                       Iodized salt      0.34%                                                       25% choline chloride                                                                            0.13%                                                       Vitamin B.sub.12  0.10%                                                       Manganese sulfate 0.02%                                                       Vitamin mix       0.06%                                                       ______________________________________                                    

Swine feed from weanling to fattening or finishing rations may besupplemented. Swine eat from about 2 lb. of ration per day (for a 25 lb.pig) to 9 lb. per day (for a 150 lb. pig). Most rations are comprised ofa corn base supplemented with legume silage, wheat bran, oats, barley,molasses or a protein supplement.

Poultry feeds comprise starter rations, broiler rations and layingrations. The rations are usually based on ground corn, corn meal orsoybean meal. The broiler rations, often, contain high energysupplements such as added fats, proteins and vitamins. Turkey rationsare similar, but comprise only a starting ration and a growing ration.Chickens or pheasants eat from 0.03-0.3 lbs. of feed per day, turkeystwice that much. Estimated intake of feed is dependent on the weight andage of the meat producing animal.

The active ingredients selected from the group consisting of AAD 216complex, AAD 216A, AAD 216B, AAD 216C or a mixture thereof are mixeduniformly with such feed rations to give supplemented rations which are,then fed as to custom, which is, most often, ad libitum. Conveniently,to do this, a premix of the supplemental growth promotant of thisinvention, optionally combined with or without other supplements knownto this art such as an anthelmintic, a nitrogen source or an antibiotic,for example, virginiamycin or oxytetracycline is prepared by themanufacturer for sale to the formulators or feed lot operators. Theconcentration of the active ingredients selected from the groupconsisting of AAD 216 complex, AAD 216A, AAD 216B, AAD 216C or a mixturethereof in the premix is usually from 5-75% by weight or a concentration100-2000 times greater than that in the complete feed ration. The premixform may be liquid or solid. Premix vehicles are corn oil, cottonseedoil, molasses or distillers solubles to form a liquid premixpreparation. Sucrose, lactose, corn meal, ground corn, flour, calciumcarbonate or soybean meal are often used as bases for solid premixpreparations. The premix composition is, then, mixed uniformly withwhole ration which is fed to the target animal. Such premix compositionsare included in the term "feed compositions" as used herein.

The concentration of the active ingredients selected from the groupconsisting of AAD 216 complex, AAD 216A, AAD 216B, AAD 216C or a mixturethereof in the complete ration is a nontoxic but active quantity chosen,for example, from a range of about 1-1000 parts of active ingredient byweight per million parts of whole feed (ppm) or about 2-115 grams perton. Advantageously, a nontoxic quantity of active ingredient is chosenfrom the range of 10-50 ppm.

The method of this invention comprises feeding to monogastric orruminant, meat or milk producing animals, especially beef and dairycattle, sheep, swine and poultry, an effective growth promoting butnontoxic quantity of an active ingredient selected from the groupconsisting of AAD 216 complex, AAD 216A, AAD 216B, AAD 216C or a mixturethereof. Other monogastric animals whose digestive tract also featuresfermentation in a cecum or cecum-like chamber are rabbits and horses.

The supplemented feed rations, described above, are presented to theanimal by methods known to the art. Ad libitum feeding in the pasture,pen or growing shed is most convenient to increase the growth andmilking rate of the animal and to increase the feed efficiency of theoperation.

The following examples are illustrative of the production, isolation andpurification of the antibiotics of the present invention and are nottherefore to be considered in limiting the present invention asdescribed in the claims appended hereto.

The nutrient media employed in the following examples have thecompositions listed below. The yields for the fermentations wereobtained by analytical HPLC by comparison to quantitated authenticsamples.

Medium 13 (H) was used for the seed growth of SK&F-AAD 216 throughoutthe experiments. Ingredients of medium 13H are: starch, 15 g/l; sucrose,5 g/l; dextrose, 5 g/l; HY-soy, 7.5 g/l; corn steep liquor, 5 g/l; K₂HPO₄, 1.5 g/l; NaCl, 0.5 g/l; CaCO₃, 1.5 g/l; and Mineral "S", 5 ml/l;pH to 7.0.

Mineral "S" has the following compositions: ZnSO₄ 7H₂ O, 2.8 g/l;Fe(NH₄)₂ HC₆ H₅ O₇, 2.7 g/l; CuSO₄.5H₂ O, 0.125 g/l; MnSO₄.H₂ O, 1.0g/l; CoCl₂.6H₂ O, 0.1 g/l; Na₂ B₄ O₇.H₂ O, 0.09 g/l; and Na₂ MoO₄.2H₂ O,0.05 g/l.

The ingredients of medium V-2 are: soybean flour, 15 g/l; beet molasses,10 g/l; glucose, 10 g/l; Estrasen 4 (methyl oleate), 10 g/l; and NaCl,0.3 g/l to a pH of 7.2.

EXAMPLE 1 First Seed Stage

The entire growth of a slant of K. aridum was suspended in 10 ml ofsterile water and aseptically innoculated into a 4 l. aspirator bottlecontaining 500 ml of medium 13 H. After incubation on a rotatory shaker,250 rpm, at 28° C. for 3 days, the mature culture was then employed toinnoculate the 14 l. glass vessel fermentor of Example 2 to produce asecond seed stage.

EXAMPLE 2 Second Seed Stage

A 14 l. glass vessel fermentor (New Brunswick Model 19) with 10 l ofsterile medium 13H was aseptically inoculated with 500 ml of vegetativeculture from Example 1. The vessel was operated according to thefollowing schedule:

Agitation: 400 rpm from 0-72 hr.

Aeration: 0.4 v/v/m* from 0-72 hr.

Temp.: 28° C.

Five liters of the resultant vegetative culture was then employed toinoculate the 75 l. fermentor of Example 3 to produce a third seedstage.

EXAMPLE 3 Third Seed Stage

A 75 l. fermentor (Chemapec) was run in similar fashion to Example 2.Five liters of the vegatative culture from Example 2 was used toinoculate the 75 l. fermentor containing 50 l. of medium 13H. Thefermentation apparatus was operated as follows:

Agitation: 250 rpm from 0-72 hr.

Aeration: 0.4 v/v/m from 0-72 hr.

Temp.: 28° C.

Fifty liters of the resultant vegetative culture was then employed toinoculate the 750 l. of Example 4 to produce the desired AAD 216complex.

EXAMPLE 4 Production of AAD 216 Complex

A 750 l. fermentor (ABEC) was operated in a similar fashion to Example3. Fifty liters of the vegetative culture of Example 3 was used toaseptically inoculate the 750 l. fermentor containing 600 l. of mediumV-2. The fermentor was operated as follows:

Agitation: 120 rpm from 0-168 hr.

Aeration: 0.3 v/v/m from 0-168 hr.

Temp.: 26° C.

The fermentation broth contained the AAD 216 complex which was composedof the individual factor antibiotics as follows:

AAD 216A=50 μg/ml;

AAD 216B=60.8 μg/ml; and

AAD 216C=43.5 μg/ml.

EXAMPLE 5 Production of AAD 216 Complex

A 450 l. fermentor (Chemapec) was operated in a similar fashion toExample 4. Thirty liters of vegetative culture (third seed stage)produced according to Example 3 was used to aseptically inoculate the450 l. fermentor containing 300 l. of medium V-2. The fermentor wasoperated as follows:

Agitation: 120 rpm from 0-168 hr.

Aeration: 0.4-0.5 v/v/m

Temperature: 26° C.

The fermentation broth contained the AAD 216 complex which was composedof the individual factor antibiotics as follows:

AAD 216A=46.2 μg/ml

AAD 216B=75 μg/ml

AAD 216C=48 μg/ml.

EXAMPLE 6 Isolation of AAD 216 Complex

Whole fermentation broth (600 l.) from Example 4 was clarified by rotarydrum filtration (Komline-Sanderson, Laboratory Scale Model) using filteraid (Hyflo Supercel, Johns-Manville Products Corp.) at existing broth pH(pH 7.7-8.2). The broth filtrate (420 l.) was chilled to 4° C. by batchtreatment of 100 L. volumes in a vat placed in a cold bath (methanol/dryice). The chilled broth filtrate was then precipitated by slow additionof concentrated hydrochloric acid, with mixing, to pH 3.0. The resultingprecipitate was recovered by rotary vacuum filtration using filter aidas previously described. The filter aid-product precipitate cake wasextracted by mixing with deionized water (55 l.) and adjusting to pH 7.0for 10 minutes. The aqueous extract thus obtained was filtered throughWhatman #4 filter paper to remove the filter aid. The filtrate soobtained was applied to two XAD-7 resin columns (8.5×110 cm.) at a flowrate of 0.5 vols./hr. After washing with 8 volumes of deionized water(pH 7.0) the desired AAD 216 complex was recovered by elution withaqueous methanol (50-100%). The methanolic eluate(s) containing thedesired AAD 216 complex was concentrated by evaporation using a risingfilm evaporator at 35° C. The resulting aqueous concentrate wasfreeze-dried on a Virtis shelf lyophilizer, yielding 85.1 g of AAD 216complex.

This AAD 216 complex was chromatographed on Whatman Partisil®Prep 40ODS-3 (1 kg) in a Jobin Yvon Chromatospac-Prep 100 (step gradient 15 to33 percent acetonitrile-0.1M pH 3.2 phosphate buffer) to yield anenriched AAD 216 complex which by analytical HPLC contained 1.88 g. ofAAD 216A, 2.6 g. of AAD 216B and 2.8 g of AAD 216C.

EXAMPLE 7 Isolation of AAD 216A, AAD 216B and AAD 216C

A sample of the enriched AAD 216 complex isolated according to Example 6(38 g. non-desalted) which by analytical HPLC contained 0.42 g. of AAD216A, 1.39 g. of AAD 216B and 0.08 g. of AAD 216C, was dissolved in oneliter 15% acetonitrile in 0.1M pH 6 phosphate buffer and the pH adjustedto 6.3. This solution was loaded onto a Waters Prep 500®HPLC with acolumn of Whatman Partisil®40 (ODS-3) (50 cm.×4.8 cm) at a flow rate of200 ml/minute. The column was then eluted using the following gradient:

(1) 20% aqueous acetonitrile and buffer solution until polar material(ultraviolet detector at 210 nm) was eluted;

(2) 24% acetonitrile buffer solution until AAD 216A was eluted;

(3) 26% acetonitrile buffer solution until AAD 216B was eluted;

(4) 28% acetonitrile buffer solution until AAD 216C was eluted; and

(5) 50% aqueous acetonitrile.

The appropriate fractions which were eluted from the HPLC column werethen desalted as described below to afford the pure individualantibiotics: AAD 216A, 0.25 g; AAD 216B, 1.05 g; and AAD 216C, 0.06 g.

The desalting procedure involved a pooling of the appropriate fractionsfrom the HPLC and removal of the acetonitrile at reduced pressure. Theresulting aqueous samples were loaded onto an XAD-7 resin column andeluted with deionized water until the conductivity of the outflow wasless than 25 micro MHO. The column was then eluted with aqueousacetonitrile (40-60%) and the eluant lyophilized to afford the desiredproducts.

EXAMPLE 8 Isolation of Enriched AAD 216 Complex

A 10 gram sample of AAD 216 complex isolated according to the XAD-7protocol of Example 6 which contained 0.69 g AAD 216A, 0.23 g AAD 216Band 0.21 g. AAD 216C (HPLC analysis) was dissolved 17.5 percentacetonitrile and 0.1M pH 6 phosphate buffer solution. The pH of thesolution was adjusted to 6.3 and this solution was loaded onto a WatersPrep 500® high pressure liquid chromatograph equipped with a 50 cm×4.8cm column packed with Whatman Partisil®Prep 40 (ODS-3). The column waseluted with 20 percent acetonitrile and buffer solution to remove polarmaterial and then eluted with 28% acetonitrile buffer solution to obtainthe enriched AAD 216 complex in the acetonitrile-buffer solution. Theacetonitrile in the eluant was removed at reduced pressure and theresulting aqueous solution was loaded onto an XAD-7 resin column andwashed with deionized water until the conductivity of the outflow wasless than 25 micro MHO. The column was then eluted with aqueousacetonitrile (40-60%) and the eluant lyophilized to afford 2.05 g.enriched AAD 216 complex which contained 0.59 g AAD 216A, 0.20 g AAD216B and 0.20 g AAD 216C.

EXAMPLE 9 Alternate Isolation Procedure

Alternately, the AAD 216 complex after XAD-7 purification waschromatographed utilizing the procedure in Example 7 to maximizerecovery affording AAD 216A, AAD 216B and AAD 216C in relatively purestate.

The AAD 216A so obtained from four individual runs (containing 3.9 g.,2.6 g and 2.2 g of AAD 216A by analytical HPLC) was combined andrechromatographed on the same HPLC isocratically eluting with 22%acetonitrile and 0.1M pH 6 phosphate buffer to afford, after desalting,4.6 g of AAD 216A from a middle cut in a highly purified state. Theother fractions were recycled for further purification.

The AAD 216B so obtained from the same individual runs (containing 2.5g, 3.7 g, 3.7 g and 1.4 g by analytical HPLC) was combined andrechromatographed on the same HPLC isocratically eluting with 26%acetonitrile and 0.1M pH 6 phosphate buffer to afford, after desalting2.7 g of AAD 216B from a middle cut in a highly purified state. Theother fractions were recycled for further purification.

EXAMPLE 10 Alternate Isolation of Enriched AAD 216 Complex

AAD 216 complex isolated according to the protocol of Example 6 whichcontained approximately 720 mg of AAD 216A, AAD 216B and AAD 216C wasdissolved in water (200 ml) and filtered. The filtrate was mixed with 70ml of an affinity chromatography sorbent of Affi-Gel®10 -D ala-D ala(633 mg D ala-D ala) in 0.02M pH 7.0 sodium phosphate buffer [seeCuatrecasas et al., Biochemistry, Vol. 11, No. 12, pp 2291-2298 (1972)for general procedure] and gently stirred for 1/2 hour. The mixture waspoured into a column and the aqueous phase eluted off. The column waswashed with: 0.02M pH 7.0 phosphate buffer (2×250 ml); 0.5M NH₄ OAc pH7.8 (2×250 ml.); H₂ O (250 ml); 0.1M pH 7.8 NH₄ OAc (250 ml); H₂ O (250ml.); 40% aqueous acetonitrile (70 ml.); 40% aqueous acetonitrile (1400ml); and 30% acetonitrile in 0.4M NaHCO₃ (2×250 ml). The 1400 ml--40%aqueous acetonitrile fraction was lyophilized to yield 607 mg of theenriched AAD 216 complex containing 224 mg AAD 216A, 133 mg AAD 216B and158 mg AAD 216C.

What is claimed is:
 1. An enriched AAD 216 complex which at a pH ofabout 6 has the following characteristics:(a) pale-white-yellow solidwhich decomposes at 300° to 350° C.; (b) an approximate elementalcomposition of 53.22 percent carbon, 6.14 percent hydrogen, 3.73 percentnitrogen and 0.28 percent ash; (c) an infrared spectrum in potassiumbromide which exhibits peaks at the following wave numbers in cm⁻¹ :3400, 2920, 1660, 1600, 1510, 1460, 1430, 1390, 1320, 1240, 1150, 1060and 1020; (d) an ultraviolet spectrum in acetonitrile:water (1:1) whichexhibits an absorption maximum at 280 nm under neutral and acidconditions with an E_(1%) =43.9 and at 301 nm under basic conditionswith an E_(1%) =56.8; (e) a positive reaction with periodate andnegative reaction with ninhydrin; and (f) soluble in H₂ O, methanol,dimethylsulfoxide and dimethylformamide and insoluble in ethanol,acetonitrile, acetone, diethylether and aliphatic hydrocarbons.
 2. AnAAD 216A antibiotic which at a pH of about 6 has the followingcharacteristics:(a) pale white-yellow solid which decomposes at 300° to350° C.; (b) an empirical formula C₈₁ H₈₂ N₈ O₃₀ Cl₄ ; (c) anapproximate elemental composition of 48.20 percent carbon, 5.01 percenthydrogen, 5.20 percent nitrogen, 6.43 percent chlorine, no sulfur ororganic phosphorus, when the water content was 10.80 percent; (d) aninfrared absorption spectrum in potassium bromide which exhibits peaksat the following wave numbers in cm⁻¹ : 3400, 2920, 1660, 1600, 1510,1460, 1430, 1390, 1320, 1300, 1240, 1150, 1060 and 1020; (e) a fast atombombardment (FAB) mass spectrum with M+H at 1787 (major cluster); (f) anultraviolet spectrum in acetonitrile:water (1:1) which exhibits anabsorption maximum at 280 nm under neutral and acidic conditions with anE_(1%) =51 and at 301 nm under basic conditions with an E_(1%) =73; (g)a carbon magnetic resonance spectrum at 90.56 MHz in CD₃ OD:D₂ O (1:9)at a pH of 8.5 which exhibits the following chemical shifts in part permillion (ppm) relative to TMS as standard: 177.7, 177.5, 175.5, 174.6,171.5, 170.8, 170.4, 170.2, 169.1, 161.8, 158.6, 157.9, 155.1, 155.0,152.5, 151.9, 151.6, 150.7, 146.0, 144.3, 141.7, 138.8, 138.3, 136.0,134.6, 134.5, 133.7, 130.8, 129.8, 129.4, 128.9 128.6, 127.4, 127.0,126.2, 125.6, 125.1, 122.7, 122.3, 120.9, 119.6, 118.3, 116.4, 110.5,109.6, 108.3, 104.2, 103.3, 103.1, 100.7, 98.1, 78.4, 74.4, 73.9, 73.3,72.0, 71.6, 71.3, 70.7, 67.3, 65.6, 63.6, 62.3, 61.6, 60.2, 56.8, 56.3,55.8, 54.9, 37.2, 32.7, 32.2, 29.8, 29.6, 29.5, 26.2, 23.0 and 14.5; (h)a specific rotation [α]_(D) ²⁵ =-66° (C=0.3 in H₂ O); (i) positivereaction with periodate and negative reaction with ninhydrin (j) solublein H₂ O, methanol, dimethylsulfoxide and insoluble in ethanol,acetonitrile, acetone, diethyl ether and aliphatic hydrocarbons; and (k)pK_(a) values in acetonitrile:water (3:7) as follows: 3.0, 4.9., 7.4,8.4, 10.0 and 10.3 pK_(a) values above 10.3 not determined.
 3. An AAD216B antibiotic which at a pH of about 6 has the followingcharacteristics:(a) pale white-yellow solid with which decomposes at300° to 350° C.; (b) an empirical formula C₈₂ H₈₄ N₈ O₃₀ Cl₄ ; (c) anapproximate elemental composition of 49.67 percent carbon, 5.07 percenthydrogen, 5.19 percent nitrogen, 6.70 percent chlorine, no sulfur ororganic phosphorus, when the water content was 10.8 percent; (d) aninfrared absorption spectrum in potassium bromide which exhibits peaksat the following wave numbers in cm⁻¹ : 3400, 2920, 1660, 1600, 1510,1460, 1430, 1390, 1300, 1240, 1150, 1060 and 1020 as shown in FIG. 2;(e) a fast atom bombardment (FAB) mass spectrum with M+H at 1801 (majorcluster); (f) an ultraviolet spectrum in acetonitrile:water (1:1) whichexhibits an absorption maximum at 280 nm under neutral and acidicconditions with an E_(1%) =55 and at 301 nm under basic conditions withan E_(1%) =72.5; (g) a carbon magnetic resonance spectrum at 90.56 MHzin CD₃ OD:D₂ O(1:9) at a pH of 8.5 which exhibits the following chemicalshifts in parts per million (ppm) relative to TMS as standard: 177.9,177.4, 175.6, 174.4, 171.6, 170.9, 170.5, 170.4, 169.3, 162.4, 158.7,158.1, 155.2, 155.0, 152.6, 151.3, 150.7, 146.0, 144.3, 141.3, 138.8,138.3, 136.1, 134.7, 133.6, 130.7, 129.9, 129.8, 129.4, 129.0, 128.7,127.7, 127.5, 127.0, 126.3, 125.7, 124.7, 122.8, 122.3, 120.9, 119.9,119.6, 118.4, 116.7, 110.5, 109.6, 108.5, 104.2, 103.3, 103.1, 100.6,98.1, 78.5, 74.4, 73.9, 73.4, 72.1, 71.7, 71.2, 70.8, 67.3, 65.5, 63.7,62.3, 61.6, 60.3, 56.8, 56.2, 55.9, 55.0, 39.6, 37.3, 32.7, 30.2, 30.0,29.7, 28.4, 27.8, 26.3 and 23.3; (h) a specific rotation [α]_(D) ²⁵=-59° (C=0.1 in H₂ O); (i) positive reaction with periodate and negativewith ninhydrin; (j) soluble in H₂ O, methanol, dimethylsulfoxide andinsoluble in ethanol, acetonitrile, acetone, diethylether and aliphatichydrocarbons; and (k) pK_(a) values in acetonitrile:water (3:7) asfollows: 3.0, 4.5, 7.5, 8.5 and 9.7 pK_(a) values above 9.7 notdetermined.
 4. An AAD 216C antibiotic which at a pH of about 6 has thefollowing characteristics:(a) pale white-yellow solid with no meltingpoint to 350° C.; (b) an empirical formula C₈₃ H₈₆ N₈ O₃₀ Cl₄ ; (c) anapproximate elemental composition of 47.89 percent carbon, 5.09 percenthydrogen, 4.95 percent nitrogen, 6.39 percent chlorine; 3.69 ash, nosulfur or organic phosphorus, when the water content was 8.2 percent;(d) an infrared absorption spectrum in potassium bromide which exhibitspeaks at the following wave numbers in cm⁻¹ : 3400, 2920, 1660, 1600,1505, 1430, 1390, 1295, 1240, 1150, 1060 and 1020 as shown in FIG. 3;(e) a fast atom bombardment (FAB) mass spectrum with M+H at 1815 (majorcluster); (f) an ultraviolet spectrum in acetonitrile:water (1:1) whichexhibits an absorption maximum at 280 nm under neutral and acidconditions with an E_(1%) =51 and at 301 nm under basic conditions withan E_(1%) =75; (g) a carbon magnetic resonance spectrum at 90.56 MHz inCD₃ OD:D₂ O(1:9) at a pH of 8.5 which exhibits the following chemicalshifts in parts per million (ppm) relative to TMS as standard: 177.9,177.2, 175.7, 173.6, 171.5, 170.8, 170.6, 170.2, 169.3, 158.6, 157.8,155.2, 154.9, 152.7, 151.9, 150.9, 146.0, 144.3, 141.3, 138.8, 138.4,136.0, 134.9, 134.7, 133.8, 130.8, 129.9, 129.8, 129.3, 129.1, 127.7,127.5, 127.0, 126.4, 125.3, 122.7, 121.0, 119.7, 118.3, 116.1, 110.4,109.6, 108.1, 104.1, 103.2, 101.5, 98.0, 78.6, 74.6, 73.9, 73.4, 72.1,71.7, 71.3, 70.3, 67.3, 65.1, 63.7, 62.5, 61.6, 60.3, 56.8, 56.3, 55.3,54.9, 39.7, 37.4, 32.6, 32.3, 30.5, 30.4, 30.2, 29.9, 28.6, 28.1, 26.4,23.4 and 22.0; (h) a specific rotation [α]_(D) ²⁵ =-50.6 (C=0.6 in H₂O); (i) positive reaction with periodate and negative reaction withninhydrin; (j) soluble in H₂ O, methanol, dimethylsulfoxide andinsoluble in ethanol, acetonitrile, acetone, diethylether and aliphatichydrocarbons; and (k) pK_(a) values in acetonitrile:water (3:7) asfollows: 3.0, 4.2, 7.2, 8.2, 9.9 and 10.3 pK_(a) values above 10.3 notdetermined.
 5. A process for the manufacture of an antibiotic complexaccording to claim 1 by the culturing of Kibdelosporangium aridumShearer gen. nov., sp. nov. ATCC 39323 microorganisms or mutants thereofin an aqueous nutrient medium containing assimilable sources of nitrogenand carbon under submerged aerobic conditions until a substantial amountof the complex is produced and isolating the complex so produced.
 6. Aprocess according to claim 5 which further comprises the isolation ofAAD 216A.
 7. A process according to claim 5 which further comprises theisolation of AAD 216B.
 8. A process according to claim 5 which furthercomprises the isolation of AAD 216C.
 9. A process according to claim 5wherein the culturing of the microorganism occurs at a temperature of15° to 42° C.
 10. A process according to claim 9 wherein the temperatureis 26° to 28° C.
 11. A process according to claim 10 wherein thetemperature is 26° C.
 12. A process according to claim 5 wherein theculturing of the microorganism continues from 3 to 8 days.
 13. Anantibacterial composition comprising an antibacterial effective amountof an enriched AAD 216 complex of claim 1 and a pharmaceuticallyacceptable carrier.
 14. An antibacterial composition comprising anantibacterial effective amount of AAD 216A of claim 2 and apharmaceutically acceptable carrier.
 15. An antibacterial compositioncomprising an antibacterial effective amount of AAD 216B of claim 3 anda pharmaceutically acceptable carrier.
 16. An antibacterial compositioncomprising an antibacterial effective amount of AAD 216C of claim 4 anda pharmaceutically acceptable carrier.
 17. An animal feed compositioncomprising a nontoxic amount of an enriched AAD 216 complex of claim 1which is effective in increasing the growth rate and feed efficiency ofmeat or milk producing animals in an animal feed.
 18. An animal feedcomposition comprising a nontoxic amount of AAD 216A of claim 2 which iseffective in increasing the growth rate and feed efficiency of meat ormilk producing animals in an animal feed.
 19. An animal feed compositioncomprising a nontoxic amount of AAD 216B of claim 3 which is effectivein increasing the growth rate and feed efficiency of meat or milkproducing animals in an animal feed.
 20. An animal feed compositioncomprising a nontoxic amount of AAD 216C of claim 4 which is effectivein increasing the growth rate and feed efficiency of meat or milkproducing animals in an animal feed.
 21. An animal feed premixcomposition comprising a premix vehicle and from 5-75% by weight of anenriched AAD 216 complex of claim
 1. 22. An animal feed premixcomposition comprising a premix vehicle and from 5-75% by weight of AAD216A of claim
 2. 23. An animal feed premix composition comprising apremix vehicle and from 5-75% by weight of AAD 216B of claim
 3. 24. Ananimal feed premix composition comprising a premix vehicle and from5-75% by weight of AAD 216C of claim 4.